The present invention relates to the field of organ transplantation, more particularly to methods for detecting organ transplantation failure.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference in their entirety for all that they disclose, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Transplant Rejection: Transplant rejection occurs when a transplanted organ, tissue, or stem cell is not accepted by the body of the transplant recipient. This is explained by the concept that the immune system of the recipient attacks the transplanted organ or tissue. This is expected to happen, because the immune system's purpose is to distinguish foreign material within the body and attempt to destroy it (http://en.wikipedia.org/wiki/Transplant_rejection).
Although immunosuppressive drugs are used to prevent organ rejection, organ and tissue transplantation would almost always cause an immune response and result in destruction of the foreign tissue. Thus, the dying cells of the foreign tissue releases their DNA and RNA into peripheral blood.
Current Methods in Detection of Transplant Rejection: Currently, transplant rejection can be found by symptoms and signs that the organ isn't functioning properly, such as elevated creatinine or less urine output with kidney transplants, shortness of breath and less tolerance to exertion with heart transplants, and yellow skin color and easy bleeding with liver transplants. A biopsy of the transplanted organ can confirm that it is being rejected. When organ rejection is suspected, one or more of the following tests may be performed prior to organ biopsy, such as abdominal CT scan, chest x-ray, heart echocardiography, kidney arteriography, kidney ultrasound, and lab tests of kidney or liver function. Although the above examinations are available, it is still difficult to detect early stage, particularly very early stage, transplant rejection.
Limits of PCR-Based Technologies: PCR-based methods, such as allele-specific PCR (Newton et al., 1989; Nichols et al., 1989; Sommer et al., 1989; Wu et al., 1989), peptide nucleic acid (PNA) clamping blocker PCR, allele-specific competitive blocker PCR, can typically detect a copy of the point mutation in no more than 102 copies of the wildtype genome, otherwise they cause false positives (Parsons and Heflich, 1997).
In addition to above potential false positives, it is difficult for PCR to detect a single copy of mutations because of primer dimers and false priming sites, causing possible false negatives.
PAP Technology: Pyrophosphorolysis-Activated Polymerization (PAP) is a new nucleic acid amplification technology that has surprising properties for nucleic acid amplification (Liu and Sommer, 2004). For example, its amplification selectivity, or signal to noise ratio, is so extremely high that it can detect a single copy of DNA mutant molecule in 1 billion of almost identical wild type molecules. This level of selectivity is over 1,000,000 times more than that of PCR or any other technologies. In addition, its sensitivity or the detectable smallest copy number of the target molecule can reliably arrive at a single copy level. This level of sensitivity is over 100 times more than that of PCR technology.
It is desired to develop new techniques for monitoring early transplantation failure in transplant donors.